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o DOI: 10.4172/1948-5956. J

ISSN: 1948-5956 Cancer Science & Therapy

Research Article Open Access Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard in Human Glioblastoma Cell Lines Gangadhara R Sareddy1#, Divya Kesanakurti2#, Pulugurtha Bharadwaja Kirti3* and Phanithi Prakash Babu1* 1Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, India 2Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL, USA 3Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India #Both the authors contributed equally

Abstract Glioblastoma represents the deadliest tumors of the central nervous system and their bleak prognosis remains unchanged over the past few decades and requiring new treatments that curb the tumor progression. Cancer cells possess high reactive oxygen species (ROS) levels when compared to their normal control counterparts. We have earlier characterized the ROS-scavenging annexin protein, BjAnn1 from Brassica juncea and demonstrated its peroxidase activity. Since are widely conserved across evolutionary lines with involvement in stress alleviation, we attempted to verify the effect of ectopic expression of ROS-scavenging BjAnn1 in human glioblastoma cell lines. The multiple sequence alignment revealed that BjAnn1 showed homology to human annexin A13 (73.18%), (70.66%), annexin A3 (72.53%) and annexin A8 (70.03%). Stable expression of BjAnn1 in U251 and U87 cells inhibited proliferation, migration and invading abilities in correlation with significant decrease in ROS levels in comparison to the empty vector (EV)-expressing controls. Multiple -profiling using cDNA- PCR arrays revealed a prominent transcriptional upregulation of oxidative stress responsive including CCS, CYB, DUOX2, FOXM, GSS, MBL2, MT3, MTL5, NME5, PXDN, SOD2 and SOD3 in BjAnn1-expressing glioblastoma cells. Western blotting confirmed the significant increase in the expression of antioxidant enzymes SOD2 (MnSOD) and SOD3 (Cu/ZnSOD) in BjAnn1-expressing cells. We also observed a significant inhibition in nuclear translocation of p65 and p50, NF-κB reporter activity and expression of NF-κB-target genes in BjAnn1 expressing cells. In summary, our study shows a prominent tumor-suppressive role of ROS-scavenging BjAnn1 in glioblastoma cell lines suggesting it as a novel candidate for efficient gene therapy in glioblastoma.

Keywords: Glioblastoma; Plant annexin; Oxidative stress; activity of plant annexins and this activity was primarily attributed to Antioxidant enzymes; Reactive oxygen species the presence of sulfur cluster in the first annexin domain and conserved His40 residue at the N-terminal region which is homologous to haem- Introduction binding domain of horse-radish peroxidase, however, subsequent Oxidative stress is caused due to the redox imbalance and excessive site-directed mutagenesis studies confirmed that His40 is not essential generation of reactive oxygen species (ROS) including superoxide (O2-), for maintaining the annexin peroxidase activity [18-20]. Conversely, - animal annexins lack His40 residue and were not reported to display hydrogen peroxide (H2O2) and hydroxyl radical (OH ) as a result of various metabolic enzymatic processes in cells [1-3]. Although basal levels of any peroxidase activity until now. Upon heterologous expression, ROS are required to regulate various signaling pathways in proliferating AnnAt1 from Arabidopsis thaliana displayed peroxidase activity and cells, excessive ROS accumulation induces deleterious modifications rescued H2O2-sensitive E. coli ∆OxyR mutant and mammalian cells on essential macromolecules such as lipids, and DNA leading from oxidative stress-induced cell death [21-23]. Our earlier studies to cellular damage [3]. To cope with high ROS, cells employ various demonstrated that the Brassica juncea annexin 1 protein (BjAnn1), defense mechanisms using antioxidant enzymes including catalases, the close homolog of AnnAt1, also exhibits in vitro peroxidase activity peroxidases and superoxide dismutases [3-5]. However, several studies [24]. The transgenic overexpression of BjAnn1 in tobacco and cotton demonstrated the critical role of increased ROS levels in the tumor conferred tolerance towards multiple stresses including oxidative stress initiation and progression by increasing cell proliferation, migration by scavenging stress-induced ROS formation [24-25]. and invasion [6]. The characteristic anchorage-independent growth and resistance towards anoikis-mediated cell death was ascribed to the high ROS levels and constant oxidative stress in tumor cells when *Corresponding authors: Phanithi Prakash Babu, Professor and Head, compared to their normal counterparts [7,8]. High ROS levels were Department of Biotechnology, School of Life Sciences, University of Hyderabad, also implicated in the maintenance of stem-like characteristics of Hyderabad, India-500046, Tel: 0091-40-23134584; 23013336; Fax: 0091-40- 23010120; E-mail: [email protected], [email protected] the cancer-initiating cells and anchorage-independent growth in the malignantly transformed invading cancer cells [8,9]. Pulugurtha Bharadwaja Kirti, Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, India-500046, Tel: 0091-40- Annexins are a superfamily of calcium-binding proteins that bind 23134584; 23013336; Fax: 0091-40-23010120 to negatively charged membrane phospholipids in cells and were Received April 27, 2013; Accepted June 20, 2013; Published June 24, 2013 reported to be conserved in eukaryotes [10-12]. Interestingly emerging Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel evidences have shown that annexins are among the genes, whose Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in expression is differentially altered in various neoplasms [13]. Annexin Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948- 5956.1000214 gene expression is differentially regulated by various stress stimuli and is reported to be involved in exocytosis, actin binding, ion transport, Copyright: © 2013 Gangadhara Reddy S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which intracellular , growth regulation, apoptosis and permits unrestricted use, distribution, and reproduction in any medium, provided inflammation, [14-17]. Several studies demonstrated the peroxidase the original author and source are credited.

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 256 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

Glioblastoma (GBM) are the most malignant, adult brain tumors clonogenic assays, EV- and BjAnn1-expressing U87 and U251 cells and account for ~80% of all primary brain tumors [26,27]. Despite (500 cells/well) were seeded in 6-well plates and allowed to grow for a modest improvement in the standard therapies, the median survival further period of 8 days. The cells were then fixed in ice cold methanol is approximately 12–15 months after diagnosis [28]. Therefore, finding and stained with 0.5% crystal violet solution to visualize the colonies. new therapeutic options against these aggressive tumors is clinically qRT-PCR and cDNA-PCR array significant. Elevated ROS has been correlated with the activation of several signaling mechanisms essential for tumor initiation and Total RNA was isolated from empty vector (pEV) and pcDNA- progression including NF-κB pathway [29]. Constitutive activation BjAnn1 expressing U251 and U87 cell lines using TRIzol® reagent and semi- of NF-κB pathway is evident in glioblastomas and is associated quantitative RT-PCR analysis were performed as described earlier [39]. with enhanced cell growth, cell cycle progression, chemoresistance, The following primers were used for semi-quantitative and quantitative migration and invasion [30-32]. Previous reports showed the anti- PCR analyses: BjAnn1 sense 5´-ATCTTGCTCTGGACTCTTGAA- proliferative roles of antioxidant enzymes in cancer cell lines by 3´and antisense 5´-TGGTGTGGTGAGCGACAT-3´and GAPDH detoxification of ROS [5,33,34]. Among these, manganese-superoxide sense 5´-GGAGTCAACGGATTTGGTCGTAT-3′ and antisense, dismutase (SOD2) was shown to be a tumor suppressor and implicated 5′-GTCTTCACCACCATGGAGAAGGCT-3′, respectively. The in oxidative stress response in various cancers [7,35,36]. Overexpression potential changes in the ROS-related gene expression profile by BjAnn1 of SOD2 and SOD3 reduced the malignant phenotype and abrogated overexpression were analyzed by performing the cDNA PCR arrays in tumor growth in gliomas [5,35]. EV-and BjAnn1-expressing U87 cells using Human Oxidative Stress and Antioxidant Defense (PAHS065) RT2 Profiler PCR Array kits Based on the significant sequence homology with human annexins, following the manufacturer’s instructions (SA Biosciences, Frederick, peroxidase and ROS-detoxifying activities of BjAnn1, the present MD, USA) as described earlier [40]. The following PCR conditions study was performed to investigate whether BjAnn1 could function were used: 1 cycle of 95°C for 10min and 40 cycles of 95°C for 15s, in human GBM cell lines across evolutionary boundaries. With the 60°C for 30s, 72°C for 30s, followed by 1 cycle of 72°C for 10min. The implication of BjAnn1 in oxidative stress, we aimed at identifying its data obtained from three independent arrays were analyzed using effect on proliferation and migration in GBM cell lines. Our results iCycler IQ version 3.1 software (Bio-Rad Laboratories, Hercules, CA, demonstrated that, BjAnn1 exhibited a novel inhibitory effect on cell USA) and Ct values were converted into fold change of expression proliferation and migration suggesting it as a potential therapeutic with 2-ΔΔCt method (ΔΔCT=ΔCT treatment [BjAnn1]−ΔCT control target in the future treatment of glioblastoma. [EV]). The gene expression changes in BjAnn1 cells in comparison to Materials and Methods EV-controls were determined by fold change values using web-based analysis software and presented in the form of a heat map (http:// Cell culture and reagents pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). Human glioblastoma cell lines U251 and U87 were obtained from Western blotting American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% FBS (Life Technologies, Carlsbad, Whole cell lysates were prepared from GBM cells in modified RIPA CA), 50 units/mL penicillin and 50 μg/mL streptomycin. Specific buffer (150 mM NaCl, 50 mM Tris-HCl, 50 mM NaF, 5 mM EDTA, antibodies against p50, p65, cyclinD1, c-Myc, SOD2, SOD3, Lamin 0.5% [wt/vol] sodium deoxycholate and 1% Triton X-100) containing B, GAPDH, β-actin and HRP-conjugated secondary antibodies were phosphatase and protease inhibitors [37]. The cytosolic and nuclear purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa extracts were prepared as described earlier [38]. Lysates were run on Fluor® conjugated secondary antibodies were obtained from Life SDS-PAGE followed by Western blotting and immunoreactivity was Technologies (Carlsbad, CA). Specific epitope-based antibody of visualized using enhanced chemiluminescence detection kit. BjAnn1 was custom synthesized (IMGENEX, India). Wound-healing migration and Transwell-Matrigel invasion Plasmid construction and stable transfection assays The full-length coding region of Brassica juncea Annexin 1 Wound-healing cell migration and Transwell-matrigel invasion (BjAnn1) was amplified using sequence-specific primers, BjAnn1 assays were performed as described earlier [40]. Briefly, the EV- sense 5´-GCGAATTCCACCATGATGGCGACTCT-3´, antisense and BjAnn1-expressing U251 and U87 cells were cultured in 6-well 5´-TCGGATCCTTAAGCATCATCTTCACCG-3´ and cloned in plates (2×105 cells/well) overnight and a straight scratch was made in mammalian expression vector pcDNA3.0 at EcoRI and BamH1 sites. individual wells using 200-μl pipette tip, which has been considered as Transfection experiments were performed with empty pcDNA vector point 0 h. After 12 hours, plates were observed for wound closure and (EV) or pcDNA-BjAnn1 in 6-hour serum-starved U251 and U87 cells average cell migration distances were measured using a microscope using lipofectamineTM 2000 following manufacturer’s instructions (Life calibrated with an ocular micrometer. For matrigel invasion assay, Technologies). Stable expression clones were selected with G418 as Transwell inserts (ThinCertTM; Greiner Bio-One, Monroe, NC) with reported earlier [37]. 8 mm pores were coated with 200 μL growth factor reduced Matrigel (50 μg/mL in serum-free medium) (Collaborative Research, Inc., Cell viability and clonogenic assay Boston, MA) for 2-3 h by placing in 37°C incubator. About 500 μL of Cell viability was determined by MTT assay in empty vector, pEV- cell suspension (1×104 cells/well) was added in each well. Invaded cells and BjAnn1-expressing cells as described earlier [38]. Briefly, U251 and through matrigel after 24 h were stained with Hema-3 and visualized U87 cells were seeded in 96-well plates (2 × 103 cells/well) in DMEM under light microscope. Percentage of cell invasion and relative medium containing 10% serum. After incubation for 72 h, growth migration distance values were represented as mean ± SE obtained inhibition was determined by using traditional MTT assays. For the from three experimental replicates.

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 257 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

ROS measurement and Immunocytochemistry Sequence analyses revealed that the mustard annexin BjAnn1 shows the most striking amino acid sequence homology up to 73.18% with Oxidative stress and superoxide content was determined using total human annexin A13 (Figure 1A). BjAnn1 also showed significant ROS/Superoxide Detection Kit following manufacturers’ instructions. sequence homology with human annexins A2 (70.66%), A3 (72.53%) In brief, the EV- and BjAnn1-expressing U251 and U87 cells were and A8 (70.03%). All annexins are characterized by the presence of a seeded in 96-well plates (1×104/well) and supplemented with 10 μM core domain made up of four similar repeats of approximately 70 amino H O for 2 hours in independent experiments as described above in 6-8 2 2 acids long. The four annexin repeats are packed to form a flattened replicates. H O -containing medium was replaced with fresh medium 2 2 disc-like structure with a slight convex surface on which the type II and and cells were further incubated for another 12-16 hours. Then cells type III Ca2+ binding loops are located and a concave surface where the were washed gently with 1× wash buffer followed by incubation in 100 -NH and -COOH termini come side-by-side [17, 45]. To analyze μl/well of ROS/Superoxide detection mix for 1 h at 37°C. Total ROS 2 the potential homology between the four conserved annexin repeats, content was determined by reading culture plates with a fluorescence we performed a domain-specific sequence alignment and generated microplate reader at 488 nm-530 nm. Green fluorescence of H DCFDA 2 sequence logos using WebLogo 3.3 program (Figure 1B). Several depicting ROS levels was also visualized under a confocal microscope. conserved and semi-conserved residues were identified in all four Immmunocytochemical analysis was performed as described earlier annexin motifs. [38]. Briefly, pEV- and BjAnn1-expressing cells were seeded in 4-well chamber slides at a density of 1×104/well and incubated overnight. Ectopic expression of BjAnn1 in human GBM cell lines Cells were fixed with 4% paraformaldehyde and permeabilized in Our earlier studies demonstrated the peroxidase activity of mustard 0.1% Triton-X100. Non-specific binding was blocked by treating the annexin (BjAnn1) and its beneficial effects in alleviating oxidative cells with 3% BSA and the cells were incubated with anti-BjAnn1 antibody (1:100 dilutions) overnight at 4°C. Cells were washed and incubated with Alexa Fluor®-conjugated secondary antibodies for A BjAnn1 AnxA13 1 hour at room temperature, washed, mounted and observed under Anx A2 Anx A3 a confocal microscope. The nuclei were counterstained with DAPI Anx A8

(4’-6-diamidino-2-phenylindole). BjAnn1 AnxA13 Anx A2 Anx A3 Reporter gene assays Anx A8

BjAnn1 Reporter gene assays were performed as described earlier [41]. AnxA13 Anx A2 Control (EV) or BjAnn1 expressing U251 and U87 cells were seeded Anx A3 Anx A8 in 6-well plates. After overnight incubation, the cells were transiently BjAnn1 TM AnxA13 transfected with NF-κB-Luc plasmid using lipofectamine 2000 for 6 Anx A2 Anx A3 h. After 48 h of transfection, cells were further treated with TNF-α (10 Anx A8 BjAnn1 ng/ml) and incubated for an additional period of 12 h. Each transfection AnxA13 Anx A2 was carried out in triplicate and normalized with the β-gal activity Anx A3 and total protein concentration. Luciferase activity was measured by Anx A8 BjAnn1 AnxA13 using the luciferase assay system (Promega, Madison, WI). Luciferase Anx A2 Anx A3 activity was expressed as percent of relative light units versus untreated Anx A8 B transfected cells. Annexin domain I 4.0 3.0 2.0 bits Bioinformatic analyses 1.0 0.0 5 10 15 20 25 30 35 40 45 50

The potential mammalian homologs of BjAnn1 were primarily Annexin Domain II 4.0 identified by performing BLAST with amino acid sequence and 3.0 2.0 bits multiple sequence alignment was performed by with ClustalW2 1.0 0.0 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) & Phylogeny.fr [42], 5 10 15 20 25 30 35 40 45 50 Annexin Domain III 4.0 http://www.phylogeny.fr/version2_cgi/index.cgi. Conserved annexin 3.0 2.0 bits domains in different annexin proteins were identified by Sequence 1.0 0.0 SMART [43], http://smart.embl-heidelberg.de/. Aligned individual 5 10 15 20 25 30 35 40 45 50 Annexin Domain IV domains were displayed as sequence-logo plots using WebLogo 3.3 4.0 3.0 2.0 [44], http://weblogo.threeplusone.com/create.cgi. bits 1.0 0.0 Statistical analysis 5 10 15 20 25 30 35 40 45 50 Figure 1: Sequence alignment of Brassica juncea annexin 1 (BjAnn1) with Data obtained from different sets of treatment groups were human annexins. (A) The amino acid sequences of BjAnn1 (ABB59547) and human annexins A2 (AAH23990), A3 (CAG28576.1), A8 (AAH73755.1) and statistically analyzed by one-way ANOVA with Tukey’s test. The mean A13 (CAA77578.1) were aligned using ClustalW2 and Phylogeny.fr. The blue ± SE values from at least three individual repetitions were presented and gray highlighting indicates conserved and semi-conserved substitutions, and the significant difference was denoted at p ≤ 0.01. respectively, in the multiple sequence alignment. (B) Representative four annexin domains (domain I: 28-80aa, domain II: 100-152aa, domain III: 183-232aa and Results domain IV: 259-311aa) in BjAnn1 and human annexins A2, A3, A8 and A13 were identified by SMART sequence analyses. Sequence logos were created Brassica juncea Annexin 1 (BjAnn1) is a close homolog to by subjecting the domain-specific sequence alignment as input to WebLogo 3.3 animal annexins program (http://weblogo.threeplusone.com/create.cgi). The height of each amino acid directly reflects the degree of conservation and frequency of that amino acid Annexin proteins are widely conserved in plants and animals. in the alignment.

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 258 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

stress upon overexpression in transgenic tobacco and cotton plants from oxidative stress [24,25]. GBM cells have been shown to exhibit Control GHCer Cer IL-2 IFN-γ IL-4 high ROS levels when compared to their normal counterparts and ROS A B C is essential in maintaining increased cell proliferation, migration and 7.2 21 48 invasion in tumors. In the present investigation, we aimed at verifying 4.8 14 32 µ g/ml µ g/ml pg/ml the effect of ROS-scavenging BjAnn1 expression in GBM cells. To 2.4 7 16 0.0 0 0 accomplish this, we ectopically expressed BjAnn1 in human U251 splenocytes CD4+ T cells splenocytes CD4+ T cells splenocytes CD4+ T cells and U87 GBM cells. The stable expression of BjAnn1 was confirmed by Western blotting (Figure 2A) and RT-PCR (Figure 2B). To identify D750 E 75 F 15 the sub-cellular localization of BjAnn1, immunofluorescence analyses 500 50 10 250 25 5 was performed. The BjAnn1 protein was primarily localized in the IgM/(ng/ml) IgG/(ng/ml) % CD138+ cells 0 0 0 membrane and cytoplasmic regions in the GBM cell lines. These results medium LPS LPS+IL-4 medium LPS LPS+IL-4 medium LPS LPS+IL-4 are in corroboration with the cellular localization of human annexins, +CD40L +CD40L +CD40L which possess phospholipid and F-actin-binding activity and display abundant membrane localization (Figure 2C). BjAnn1 expression inhibited proliferation, colony formation, migration and invasion in U251 and U87 GBM cells To study the role of BjAnn1 on proliferation, an MTT assay Figure 3: Ectopic expression of BjAnn1 reduces the proliferation, migration and colony formation of GBM cell lines. (A) U251 and U87 cells were transfected with was performed. In comparison to EV-treated controls, BjAnn1- control (pEV) and BjAnn1 plasmids and the proliferation was determined at 72 expressing U251 and U87 cells showed significance decrease in cell h by using the MTT assay. All data presented are the mean of 3 independent proliferation (Figure 3A). Further, clonogenic assay revealed that experiments with mean ± SE. *, p < 0.05. (B) U251 and U87 cells stably expressing control (pEV) or BjAnn1 plasmids were seeded in 6 well plates (500 cells/well) colony-forming ability of BjAnn1-expressing cells was prominently and allowed to grow for 8 days. Colonies were stained with crystal violet and inhibited in comparison to EV-controls (Figure 3B). It is well evident colonies that contain ≥50 cells were counted. All data presented are the mean of that glioma cells possess high migratory and invasive potential and 3 independent experiments with mean ± SE. *, p < 0.05. (C) U251 and U87 GBM cells were transfected with control (pEV) and BjAnn1 plasmids were subjected often metastasized into surrounding normal brain parenchyma. We scratch wound healing assay as described in materials and methods and the next determined the effect of BjAnn1 expression on migration and distance of migration was determined after 12 hours. (D) U251 and U87 cells invasion of U87 and U251 cells by performing scratch wound-healing transfected with control (pEV) and BjAnn1 plasmids were subjected to matrigel invasion assay and the percentage of invaded cells compared to control were assay and matrigel invasion assay respectively. As shown in Figure 3C, plotted on histogram. the migratory potential of BjAnn1-expressing cells was significantly decreased when compared to pEV-expressing cells. Correspondingly, the invasive potential through matrigel was significantly inhibited in invasiveness were in part attributed to the high ROS levels and ROS- U87 and U251 upon BjAnn1 expression (Figure 3D). These results dependent signaling activation in tumors. Based on the previous suggest the anti-proliferative and anti-migratory effect of BjAnn1 in reports showing peroxidase activity of BjAnn1, we next determined its GBM cells. effect on ROS levels in U87 and U251 cells. The BjAnn1-expressing cells showed a significant reduction in ROS levels compared to BjAnn1 reduced ROS levels and modulated the expression of the mock- or pEV-controls (Figure 4A). Based on this interesting oxidative stress and antioxidant defense genes result of significant ROS diminution, we attempted to analyze the Characteristic traits of deregulated cell proliferation and possible modulation in expression levels of genes responsive to ROS- dependent signaling and metabolism. To achieve this, we performed a human oxidative stress and antioxidant defense PCR array. BjAnn1 A B expressing cells showed prominent transcriptional upregulation of

Mock EV BjAnn1 Superoxide dismutase copper chaperone (CCS), Cytochrome b (CYB), EV BjAnn1 EV BjAnn1 BjAnn1 NADPH oxidase (DUOX2), Forkhead box M1 (FOXM), Glutathione GAPDH synthetase (GSS), Mannose-binding lectin (protein C) (MBL2), BjAnn1 U87 U251 U87 U251 U87 Metallothionein 3 (MT3), Metallothionein-like 5 (MTL5), Peroxidasin homolog (PXDN), Mn superoxide dismutase (SOD2) and Cu/Zn C BjAnn1 DAPI BjAnn1 DAPI extracellular superoxide dismutase (SOD3) genes, which are involved in superoxide metabolism, apoptosis, transcriptional regulation and (Figure 4B). The expression of genes upregulated in response to BjAnn1 expression was summarized in Table 1. With

U251 U87 the striking transcriptional elevation of SOD2 (MnSOD) and SOD3 (Cu/ZnSOD) in BjAnn1 expressing GBM cells, we further checked Figure 2: Confirmation of stable BjAnn1 expression in human GBM cell lines. (A) Whole cell lysates from EV- and BjAnn1-expressing cells were subjected to the protein levels. Ectopic expression of BjAnn1 significantly elevated Western blotting. Blots were re-probed with GAPDH to monitor equal loading. (B) SOD2 and SOD3 expression levels in both cell lines (Figure 4C). To Semi-quantitative RT-PCR was performed in EV- and BjAnn1-expressing U251 confirm the specificity of BjAnn1 function in alleviating ROS in GBM and U87 cells. (C) Confocal microscopy was performed as described in Materials and Methods. Representative micrographs of BjAnn1 (red) expression were cells and to rule out the off-target consequences, we treated U87 cells presented from randomly selected microscopic fields. Nuclei were counterstained with H2O2. Notably, treatment with H2O2 significantly enhanced ROS with DAPI. levels in control and EV-treated cells (Figure 4D). On the other hand,

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 259 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

A 01 02 03 04 05 06 07 08 09 10 11 12 B A +6 B Mock EV BjAnn1 C 150 D 0 120

90 BjAnn1/EV E 60 * F

30 Gene expression U87 -6 G 0 U251 150 LAYOUT 1 2 3 4 5 6 7 8 9 10 11 12 120 A ALB ALOX1 ANGPTL7 AOX1 APOE ATOX BNIP3 CAT CCLS CCS CSDE1 CYB

90 B CYGB DGKK DHCR24 DUOX1 DUOX2 DUSP1 EPHX` EPX FOXM GLRXA GPR156 GPX1

60 * C GPX2 GPX3 GPX4 GPX5 GPX6 GPX7 GSR GSS GSTZ1 GTF21 KRT1 LPO Relative ROS levels (A.U.) 30 D MBL2 MGST3 MPO MPV17 MSRA MT3 MTL5 NCF1 NCF2 NIME5 NOS2 NOX5

0 E NUDT1 OXR1 OXSR1 PDLM IPGEF1 PKP PRDX1 PRDX2 PRDX3 PRDX4 PRDX5 PRDX6 U87 F PREX1 PR3 PRNP PTGS1 PTGS2 PXDN PXDNL RNF7 SCARA SELS SEPP1 SFTPO 3 G SGK2 SIRT2 SOD1 SOD2 SOD3 SRXN1 STK26 TPO TTN TXNDC2 TXNRD1 TXNRD2

C D U251 U87 350 ** ** 300 Control

EV BjAnn1 EV BjAnn1 250 Control+H2O2 200 * EV SOD2 150 BjAnn1 SOD3 100 EV+H2O2 50 BjAnn1+H2O2 0

GAPDH Relative ROS levels (A.U.)

Figure 4: BjAnn1 exhibits ROS scavenging activity and modulates the expression of genes involved in oxidative stress and antioxidant defense. (A) U251 and U87 cells were transfected with EV- and BjAnn1 plasmids and after 48 h cells were seeded in 96-well plates (1×104/well) and ROS levels were determined using total ROS/ Superoxide Detection Kit following manufacturers’ instructions. (B) Potential changes in the multiple gene expression profile in BjAnn1-expressing U87 cells were determined in comparison to EV-treated cells using Human Oxidative Stress and RT2 Profiler Human Antioxidant Defense arrays as explained in Materials and Methods. The relative fold changes values of gene expression in BjAnn1-expressing cells in comparison to EV-treated controls were obtained from three experimental replicates and represented in the form of a heat map. (C) Whole cell lysates were prepared from control (EV) and BjAnn1 expressing U251 and U87 cells and the expression levels of SOD2 and SOD3 were determined by western blotting. (D) U87 cells transfected with EV- and BjAnn1 plasmids were seeded in 96-well plates (1×104/well) and

treated with 10 μM H2O2 for 2 hours, then fresh medium was added and further incubated for another 12-16 hours. Cells were incubated in 100 μl/well of ROS/Superoxide detection mix for 1 h at 37°C. Total ROS content was determined by reading culture plates with a fluorescence microplate reader at 488 nm-530 nm.

Gene NCBI REFSEQ Description Fold change: BjAnn1/EV

AOX1 NP_001150.3 Aldehyde oxidase 1 3.13 CCS NP_005116.1 Superoxide dismutase copper chaperone 5.03 CYB NP_536855.1 Cytochrome b 4.25 DGKK NP_001013764.1 Diacylglycerol kinase, kappa 3.51 DUOX2 NP_054799.4 NADPH oxidase 4.28 DUSP1 NP_004408.1 Dual specificity phosphatase 1 3.23 FOXM NP_001230017.1 Forkhead box M1 4.04 GPR156 NP_001161743.1 G protein-coupled receptor 15 3.43 GPX1 NP_000572.2 Glutathione peroxidase 1 3.22 GPX2 NP_002074.2 Glutathione peroxidase 2 3.97 GPX3 NP_002075.2 Glutathione peroxidase 3 3.26 GSR NP_000628.2 Glutathione reductase 3.96 GSS NP_000169.1 Glutathione synthetase 4.95 GSTZ1 NP_001504.2 Glutathione S-transferase zeta 1 3.05 MBL2 NP_000233.1 Mannose-binding lectin (protein C) 2, soluble 4.20 MPO NP_000241.1 Myeloperoxidase 3.16 MPV17 NP_002428.1 MpV17 mitochondrial inner membrane protein 3.55 MSRA NP_001129142.1 Methionine sulfoxide reductase A 3.75 MT3 NP_005945.1 Metallothionein 3 5.43 MTL5 NP_004914.2 Metallothionein-like 5, testis-specific (tesmin) 6.34 NME5 NP_003542.1 NME/NM23 family member 7.74 PKP NP_000290.2 Plakophilin 1 3.59 PRDX2 NP_005800.3 Peroxiredoxin 2 3.44 PRDX3 NP_006784.1 Peroxiredoxin 3 3.03 PREX1 NP_065871.2 PtdIns(3,4,5)-dependent Rac exchanger 1 3.14 PR3 NP_002768.3 Proteinase 3 3.23 PXDN NP_036425.1 Peroxidasin homolog 5.33 SELS NP_060915.2 Selenoprotein S 7.04 SIRT2 NP_001180215.1 Sirtuin 2 3.72 SOD2 NP_000627.2 Mn superoxide dismutase 6.42 SOD3 NP_003093.2 Extracellular superoxide dismutase [Cu-Zn] 3.94 Table 1: Modulation of oxidative stress response genes in BjAnn1-expressing cells. Total RNA was isolated from EV- and BjAnn1-stable expressing cells and cDNAs were subjected to Human Oxidative and Antioxidant Defense PCR array (PAHS65) (SABiosciences, Valencia, CA) using real-time PCR. The fold change values of gene expression were determined in BjANN1 cells in comparison to EV-treated controls in three individual experiments using web-based software (http://www.sabiosciences. com/pcrarraydataanalysis.php).

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 260 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

BjAnn1 expression significantly reversed and decreased the H2O2- Discussion induced ROS levels. These results clearly show the ROS-scavenging Despite using the advanced multimodal treatments, GBM remain activity of BjAnn1 and suggest its role in alleviating oxidative stress in to be the deadliest among the tumors of the central nervous system. GBM cells. Since the prognosis remains poor in GBM, there is an imperative need BjAnn1 attenuated the NF-κB nuclear translocation and its to develop novel and alternative therapeutic regimens in their control. activation Recently, increasing evidences suggest the possible functions of plant genes across evolutionary boundaries in the potential therapeutic Deregulated NF-κB signaling is a hallmark in GBM progression. applications in mammalian systems in vitro and in vivo [37,46]. Earlier NF-κB is known to be activated in response to ROS and critical in studies demonstrated the novel roles of plant proteins in the regulation the maintenance of tumor cell proliferation and migration. With the of cell proliferation when heterologously expressed in a variety of cancer compromised ROS levels upon BjAnn1 expression in GBM cells, we cells [22,23,46]. Recently, we showed that expression of mammalian next proceeded to check the levels of NF-κB subunits, p65 and p50. IκB homologue, NPR1 (non-expressor of pathogenesis related protein As shown in Figure 5A, the TNF-α induced p50 and p65 nuclear 1) from mustard in GBM cells inhibited their proliferation via the translocation was significantly reduced in BjAnn1 expressing cells inhibition of NF-κB pathway [37]. The Thymidine kinase 1 from compared to pEV-control cells. In contrast, the cytoplasmic p65 levels Tomato exhibited tumor suppressive role when expressed in human were high in BjAnn1 expressing cells compared to pEV-control cells. GBM cells and enhanced sensitivity to cytotoxic agents [46]. In line To further study whether BjAnn1 interfered with the NF-κB pathway with these reports and based on our earlier experiments showing the activation, we performed gene reporter assays. As shown in Figure 5B, in vitro peroxidase activity of BjAnn1, we attempted to verify the the BjAnn1 overexpression significantly reduced the NF-κB reporter effect of BjAnn1 expression in human GBM cell lines. We showed activity compared to pEV-control cells. Further, we also checked the that overexpression of BjAnn1 reduced proliferation, migration and expression of proliferation markers such as cyclin D1 and c-Myc, the invasion of GBM cells and reduced the ROS generation with the direct transcriptional target genes of NF-κB. The expression levels of enhanced expression of antioxidant superoxide dismutases, SOD2 and cyclin D1 and c-Myc were down regulated in BjAnn1 expressing GBM SOD3. Further, BjAnn1 overexpression inhibited the activation of NF- cells compared to control GBM cells (Figure 5C). These results clearly κB pathway in GBM cells. demonstrated that the expression of BjAnn1 inhibits the GBM cell The Brassica Juncea Annexin 1 (BjAnn1) expression in tobacco proliferation by interfering with NF-κB pathway activation. and cotton conferred oxidative stress tolerance and increased disease

A C

EV EV+TNF-αBjAnn1 BjAnn1+TNF-α EV EV+TNF-αBjAnn1 BjAnn1+TNF-α p65 p65

p50 p50 EV BjAnn1 EV BjAnn1 LaminB GAPDH CyclinD1 C-Myc p65 p65 Actin U251 U87 U87 U251 p50 p50 LaminB GAPDH Nuclear Cytoplasmic

NF-kB Luc B NF-kB Luc U251 7 U87 5 4,5 6 4 5 3,5 3 4 2,5 * 3 * 2 1,5 2 1 1 values (fold)

0.5 values (fold) 0 0 Relative luciferase Relative luciferase

Control BjAnn1 Control BjAnn1

Control+TNF-α BjAnn1+TNF-α Control+TNF-α BjAnn1+TNF-α Figure 5: BjAnn1 attenuated the nuclear translocation of NF-κB and reduces its target gene expression. (A) Cytoplasmic and Nuclear lysates were prepared from U251 and U87 cells that are stably expressing control (EV) and BjAnn1 plasmids and stimulated with or without TNF-α for 30 min and subjected to Western blot analysis with p65 and p50 antibodies. GAPDH and Lamin B were used as loading controls for cytoplasmic and nuclear fractions respectively. (B) U251 and U87 cells stably expressing control (pEV) and BjAnn1 were transfected with the NF-κB-Luc reporter and after 48 h cells were stimulated with TNF-α and the luciferase activity was measured. All data presented are the mean of 3 independent experiments with mean ± SE. *, p< 0.05, t test. (C) Whole cell lysates were prepared from U251 and U87 cells that are stably expressing control (EV) and BjAnn1 plasmids subjected to Western blot analysis with cyclin D1 and c-Myc antibodies. β-actin used as an loading control.

J Cancer Sci Ther ISSN:1948-5956 JCST, an open access journal Volume 5(7) 256-263 (2013) - 261 Citation: Gangadhara Reddy S, Divya K, Kirti PB, Prakash Babu P (2013) Novel Antiproliferative and Antioxidant Role of BjAnn1, a Mustard Annexin Protein in Human Glioblastoma Cell Lines. J Cancer Sci Ther 5: 256-263. doi:10.4172/1948-5956.1000214

resistance [23,24]. In both the transgenic plants, ROS scavenging suppressive antioxidant enzymes, MnSOD and Cu/ZnSOD in BjAnn1 activities were demonstrated. The Oxy5/AtAnn1 protein from expressing GBM cells. It has also been shown that ROS activates the Arabidopsis thaliana displayed peroxidase activity and protected NF-κB [26] and NF-κB pathway constitutively activated in gliomas. We oxidative stress-susceptible OxyR-mutant of E.coli from H2O2- hypothesize that the reduced proliferative and migration properties induced death [21]. In another study, overexpressed Oxy5 in CHO of BjAnn1 exert partly through inhibition of NF-κB pathway. In our cells and mouse leukemic K36 cells decreased superoxide production, study we observed that the nuclear translocation of p50 and p65 was PKC activity and inhibited subcutaneous tumor growth in nude also reduced in BjAnn1 overexpressing cells. Further, NF-κB reporter mice [23]. Upon expression in TNF-sensitive HeLa D98 cells, Oxy5 gene activity and its target gene activation such as cyclin D1 and c-Myc conferred resistance to TNF cytotoxicity and decreased TNF-induced were reduced in BjAnn1 expressing GBM cells. intracellular superoxide formation [22]. It has also been shown that In summary, our study for the first time demonstrated that annexins exhibited ROS neutralizing activity [20]. These studies heterologous expression of BjAnn1 inhibits the proliferation, migration supported the antioxidant role of annexin proteins in heterologous and invasion of GBM cells by enhancing ROS scavenging activity with systems. In our present study, we ectopically expressed the BjAnn1 in concomitant activation of antioxidant enzymes and inhibition of NF- GBM cells and found that it is localized mostly to the cell membrane κB pathway. This study may initiate the development of new treatment and cytoplasm. 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